Methods and materials for conferring tripsacum genes in maize

ABSTRACT

There is provided a method for transferring Tripsacum nuclear and cytoplasmic genes into maize. The method is via a hybrid plant designated Tripsacorn (proposed botanical classification Zea indiana), produced by crossing two wild relatives of corn, Tripsacura and diploid perennial teosinte (Zea diploperennis). This invention thus relates to the hybrid seed, the hybrid plant produced by the seed and/or tissue culture, variants, routants, and modifications of Tripsacorn and the hybrid seed, the hybrid plant produced by the seed and/or tissue culture, variants, mutants, and modifications of (maize X Tripsacorn) and/or (Tripsacorn X maize). In particular this invention is directed to the ability to confer rootworm resistance, resistance to insect pests, resistance to diseases, drought tolerance, and improved standability to maize via Tripsacorn.

This application is a continuation-in-part of application Ser. No.07/613,269, filed Nov. 13, 1990, issued as U.S. Pat. No. Plant 7,977 onSep. 15, 1992 the disclosure of which is incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates generally to the field of plant breeding. Moreparticularly, it relates to a method for the production of inbred andhybrid corn with desirable characteristics including corn rootwormresistance, resistance to insect pests, resistance to diseases, droughttolerance and improved standability conferred by Tripsacum introgressionvia a bridge species called Tripsacorn.

BACKGROUND OF THE INVENTION

Plant breeding is the science that utilizes crosses between individualswith different genetic constitutions. The resulting recombination ofgenes between different lines, species or genera produces new hybridsfrom which desirable traits are selected. Methods employed to developnew varieties or species depend on whether a crop plant reproducessexually or asexually. Since maize is a sexually reproducing plant,techniques for controlled pollination are frequently employed to obtainnew hybrids.

A significant technological breakthrough in maize breeding was thediscovery that crossing inbred lines resulted in a hybrid with greatlyenhanced vigor. Inbred lines are obtained from self-pollination andselection of homozygous plants for several generations until a pure linedescended by self-pollination from an apparently true-breeding plant isobtained. The purpose of inbreeding is to fix desirable characters in ahomozygous condition in order that the line may be maintained withoutgenetic change. Inbred lines with desired traits are then crossed toproduce commercial hybrids. Yields from hybrid maize seed are muchgreater than average yields of inbreds and open-pollinated varieties.

Maize is a monoecious grass, i.e. it has separate male and femaleflowers. The staminate, i.e. pollen-producing, flowers are produced inthe tassel and the pistillate or female flowers are produced on theshoot. Pollination is accomplished by the transfer of pollen from thetassel to the silks. Since maize is naturally cross-pollinated,controlled pollination, in which pollen collected from the tassel of oneplant is transferred by hand to the silks of another plant, is atechnique used in maize breeding. The steps involved in makingcontrolled crosses and self-pollinations in maize are as follows: (1)the ear emerging from the leaf shoot is covered with an ear shoot bagone or two days before the silks emerge to prevent pollination; (2) onthe day before making a pollination, the ear shoot bag is removedmomentarily to cut back the silks, then is immediately placed back overthe ear; (3) on the day before making a pollination, the tassel iscovered with a tassel bag to collect pollen; (3) on the day ofpollination, the tassel bag with the desired pollen is carried to theplant for crossing, the ear shoot bag is removed and the pollen dustedon the silk brush, the tassel bag is then immediately fastened in placeover the shoot to protect the developing ear. Wild relatives of cropplants are an important source of genetic diversity and genes welladapted to many different stresses. The wild relatives of maize includeannual teosinte (Zea mexicana), perennial teosinte and Tripsacum. Zeadiploperennis (hereafter referred to as diploperennis), is a diploidperennial teosinte. A previously unknown wild relative of maize, it wasdiscovered on the verge of extinction in the mountains of Jalisco,Mexico in 1979. Diploperennis, like annual teosinte, is in the samegenus as maize, has the same chromosome number (n=10), and hybridizesnaturally with it. Tripsacum is a more distant relative of maize with adifferent haploid chromosome number (n=18). The progeny of (maize XTripsacum) obtained by artificial methods are all male sterile and havelimited female fertility when pollinated by maize pollen. Cytogeneticstudies of maize-Tripsacum hybrids show partial chromosome pairing andhomology between segments of Tripsacum and maize chromosomes (Maguire1961, 1963; Chaganti 1965; Gallnat 1974). In spite of strongcross-incompatibility, the fact that maize and Tripsacum chromosomes canoccasionally pair enables limited transfer of Tripsacum genes intomaize. Attempts to make the corollary cross, i.e. between Tripsacum andteosinte, however, have heretofore failed to produce viable plants(Tantravahi 1968; deWet and Harlan 1978).

Plant breeders acknowledge Tripsacum has significant potential forimproving corn by expanding its genetic diversity (Gallnat 1977; Cohenand Galinat 1984; Poehlman 1986). The limited fertility ofmaize-Tripsacum hybrids presents a significant biological barrier togene flow between these species. Successful introgression of Tripsacumgenetic material into maize heretofore has required years ofcomplicated, high risk breeding programs that involve many backcrossgenerations to stabilize desirable Tripsacum genes in maize. Accordingto Kindiger and Beckett: "Tripsacum may be expected to contain valuableagronomic characters that could be exploited for the overall improvementof maize . . . An effective procedure to transfer Tripsacum germ plasminto maize has been needed by maize breeders and geneticists for manyyears" (1990, p. 495). Beneficial traits that may be derived fromTripsacum include heat and drought tolerance (Reeves and Bockholt 1964),elements of apomixis, increased heterosis (Reeves and Bockholt 1964;Cohen and Galinat 1984), resistance to corn root worm (Branson 1971),corn leaf aphid (Branson 1972), northern and southern leaf blight,common rust, anthracnose, fusarium stalk rot and Stewart's bacterialblight (Bergquist 1977, 1981; deWet 1979).

(Zea mays X Tripsacum) plants have unreduced gametes with 28chromosomes, one set of 10 Zea chromosomes and one set of 18 Tripsacumchromosomes. There has been one report of a successful reciprocal crossof Tripsacum pollinated by maize in which embryo culture techniques wereused to bring the embryo to maturity. The plants were sterile(Farquharson 1957). This (Tripsacura X maize) plant was employed byBranson and Guss (1972) in tests for rootworm resistance inmaize-Tripsacum hybrids. When the (maize X Tripsacura) hybrid has beencrossed with either annual teosinte or diploperennis, a trigenomichybrid has been produced that has a total of 38 chromosomes; 10 frommaize, 18 from Tripsacum and 10 from teosinte. The resulting trigenomicplants were all male sterile and had a high degree of female infertility(Mangelsdorf 1974; Galinat 1986).

Transformation, a technique from molecular biology, now offersopportunity for the asexual transfer of genes that heretofore could onlybe achieved by crossing different plant strains. In order for breedersto employ gene transfer via transformation, they first have to be ableto achieve plant regeneration from calli or protoplasts. Althoughtransformation has been successfully performed in maize (Gordan-Kamm etal. 1990), there is limitation in developing transgenic maize due to thedifficulties of plant regeneration from maize protoplasts (Potrykus1990). The problem is there are very few maize lines that can besuccessfully regenerated from maize protoplasts. In order fortransformation to be useful for commercial hybrid seed production, itwill be necessary to have inbred lines amenable to the transgenicprocess that can be regenerated by tissue culture.

Rootworms, Diabrotica spp., are a serious agricultural pest. Reductionin corn yields due to corn rootworm damage ranges from 13 to 16 bushelsper acre which is approximately 10 to 13%. Costs of insecticidetreatments and crop losses are estimated at $1 billion per year(Metcalfe 1986). Rootworm larvae feed on the root system of corn forseveral weeks passing through three instars. This is the mostdestructive stage and causes reduced yields through damage to the rootsystem or indirectly from lodging which makes plants difficult toharvest. Adult beetles feed on the aerial parts of the corn plantincluding the pollen, silks and leaves (Branson et al. 1975).

Zea diploperennis is an acceptable larval host for several Diabroticaspecies. Feeding scars and leaf damage have been recorded for plantsgrowing in the wild in Jalisco, Mexico, and laboratory screeningrevealed diploperennis has no antibiotic effect on rootworm larvae(Branson and Reyes 1983). Tripsacum dactyloides, however, has been shownto exhibit a high degree of resistance to corn rootworm (Branson 1971).Screening of intergeneric hybrids between T. dactyloides and Zea maysshowed (maize X Tripsacuraum) was susceptible; whereas, (Tripsacura Xmaize) exhibited resistance (Branson and Guss 1972). The authorsproposed two explanations: (1) resistance is inherited through thecytoplasm, or (2) the genes for resistance occur on lost Tripsacumchromosomes in (maize X Tripsacum) plants.

Polyploidy refers to all natural and induced variations in chromosomenumber. Many cultivated crop species have evolved in nature aspolyploids. One way polyploid plants arise is by combining chromosomesets from two or more species which is referred to as allopolyploidy. Anallopolyploid, i.e. a plant in which the total chromosome complement oftwo other species is combined to form a fertile species hybrid, isreferred to as an amphiploid. A plant breeding method to transfer genesacross a barrier of reproduction isolation is via bridging speciesderived from an amphiploid. This type of introgressive hybridizationproduces convergence between previously more distinct species. It mayresult in the appearance of types that are new species intermediate totheir more divergent and distinct parents. Bridging species derived fromcrosses between two parents with different chromosome numbers arefrequently characterized by a new chromosome number. The change inchromosome complement and/or rearrangements in chromosome structure mayovercome the inability of chromosomes to pair that causes infertilityand often prevents the success of wide crosses.

Two wild grasses, Zea diploperennis and Tripsacum dactyloides have beencrossed to produce a novel hybrid referred to as Tripsacorn, proposedbotanical name Zea indiana. A bridging mechanism to transfer Tripsacumgenes into maize is provided by Tripsacorn which is cross-fertile withmaize. It promises to improve corn by imparting numerous beneficialcharacteristics including pest resistance and drought tolerance.

Based on proposed taxonomic relationships between Zea and Tripsacura andthe results of prior crosses between them, the success of the crossesbetween Zea diploperennis and Tripsacura resulting in fully fertileplants with chromosome numbers of 2n=20 and 2n=18 could not have beenpredicted. The reduction in chromosome number in the interspecific crossis unexpected based on prior art. The fertility of plants resulting fromthe cross made both ways is also unexpected. Tripsacum and diploperennishave chromosomes that are very similar architecturally in length andtheir diminutive, terminal knobs that appear at one or both ends of manyof the chromosomes in both species. The small terminal knobs in thesespecies are distinct from the large internal knobs that characterize thechromosomes of corn and annual teosinte. As evidenced by cross fertilityand chromosome number, the similarities in the chromosome structure ofTripsacum and diploperennis evidently promote a greater degree ofpairing and enable the unexpected success of this cross.

The unexpected fertility of this hybrid, and its cross-fertility withmaize, is of great value because it conveys opportunity for directlycrossing with maize. Tripsacorn provides a mechanism for importingTripsacum genes into maize in one generation by natural breedingtechniques. Since Tripsacum is the female parent in this cross, itprovides unique opportunity for transferring Tripsacum cytoplasmic genesinto maize.

Insect resistance derived from crossing Tripsacorn with maize has beendemonstrated experimentally. In a series of bioassays, seedlings from(maize X Tripsacorn) infested with western corn rootworm, Diabroticavirgifera Le Conte, showed clear evidence for rootworm resistance. Thiswas corroborated by comparison with maize controls and (maize X SunDance) plants, both of which were susceptible, as indicated byconsiderable root damage or death.

REFERENCES

Bergquist, R. R. 1977. Techniques for evaluation of genetic resistancein corn. North Central Regional Corn and Sorghum Disease Project,Chicago.

Bergquist, R. R. 1981. Transfer from Tripsacum dactyloides to corn of amajor gene locus conditioning resistance to Puccinia sorghi.Phytopathology 71:518-520.

Branson, T. F. 1971. Resistance in the grass tribe Maydeae to larvae ofthe western corn rootworm. Ann. Ent. Soc. Am. 64:861-863.

Branson, T. F. 1972. Resistance to the corn leaf aphid in the grasstribe Maydeae. J. Econ. Entomol. 65:195-196.

Branson, T. F. 1986. Larval feeding behavior and host-plant resistancein maize. In J. L. Krysan and T. A. Miller (ed.) Methods for the Studyof Pest Diabrotica. Springer Verlag, New York.

Branson, T. F. and P. L. Guss. 1972. Potential for utilizing resistancefrom relatives of cultivated crops. Proc. North Central Branch Entomol.Soc. Am. 27:91-95.

Branson, T. F., P. L. Guss, J. L. Krysan and G. R. Sutter. 1975. Cornrootworms: Laboratory rearing and manipulation. U.S. Dept. Agric.,ARS-NC-28. 18 pp.

Branson, T. F. and J. Reyes R. 1983. The association of Diabrotica spp.with Zea diploperennis. J. Kan. Entomol. Soc. 56:97-99.

Chaganti, R. S. K. 1965. Cytogenetic studies of maize-Tripsacum hybridsand their derivatives. Harvard Univ. Bussey Inst., Cambridge, Ma.

Cohen, J. I. and W. C. Galinat. 1984. Potential use of alien germplasmfor maize improvement. Crop Sci. 24:1011-1015.

DeWet, J. M. J. 1979. Tripsacum introgression and agronomic fitness inmaize (Zea mays L.). Proc. Conf. Broadening Genet. Base Crops, Puduc,Wageningen.

DeWet, J. M. J. and J. R. Harlan. 1978. Tripsacum and the origin ofmaize. In D. B. Walden (ed.) Maize Breeding and Genetics, John Wiley &Sons, New York.

Elseman, L. and L. Herbert, 1990. The Pantone Book of Color. Harry N.Abrams, Inc., Publishers, New York.

Farquharson, L. I. 1957. Hybridization of Tripsacum and Zea. J. Heredity48: 295-299.

Galinat, W. C. 1974. Intergenomic mapping of maize, teosinte andTripsacura. Evolution 27: 644-655.

Galinat, W. C. 1977. The origin of corn. In G. F. Sprague (ed.). Cornand Corn Improvement. Amer. Soc. Agronomy, Madison, Wis.

Galinat, W. C. 1982. Maize breeding and its raw material. In W. L.Sheridan (ed.) Maize for Biological Research. University Press, GrandForks, N.D.

Galinat, W. C. 1986. The cytology of the trigenomic hybrid. MaizeGenetics Newsletter 60:133.

Gordon-Kamm, W. 1990. Transformation of maize cells and regeneration offertile transgenic plants. Plant Cell 2:603.

Hills, T. M. and D. C. Peters. 1971. A method of evaluating postplantinginsecticide treatments for control of western corn rootworm larvae. J.Econ. Entomol. 64: 764-765.

Iltis, H. H., J. Doebley, R. Guzman M. and B. Pazy, 1979. Zeadiploperennis (Gramineae): A new teosinte from Mexico. Science 203:186-188.

Kindiger, B. and J. B. Beckett. 1990. Cytological evidence supporting aprocedure for directing and enhancing pairing between maize andTripsacura. Genome 33: 495-500.

Maguire, M. P. 1961. Divergence in Tripsacura and Zea chromosomes.Evolution 15: 393-400.

Maguire, M. P. 1963. Chromatid interchange in allodiploidmaize-Tripsacum hybrids. Can. J. Genet. Cytol. 5:414-420.

Mangelsdorf, P. C. 1974. Corn: Its origin, evolution and improvement.Harvard Univ. Press, Cambridge, Ma.

Metcalfe, R. L. 1986. Foreword. pp.vii-xv. In J. L. Krysan and T. A.Miller (ed.) Methods for the Study of Pest Diabrotica. Springer Verlag,New York.

Poehlman, J. M. 1986. Breeding Field Crops. 3rd ed. AVI Publ. Co., Inc.,Westport, Conn.

Potrykus, I. 1990. Gene transfer to cereals: An assessment.Biotechnology:535-542.

Reeves, R. G. and A. J. Bockholt. 1964. Modification and improvement ofa maize inbred by crossing it with Tripsacum. Crop Sci. 4:7-10.

Tantravahi, 1968. Cytology and crossability relationships of Tripsacum.Harvard Univ. Bussey Inst., Cambridge, Ma.

SUMMARY OF THE INVENTION

In one embodiment of the invention, there is provided a method forconferring Tripsacum nuclear and cytoplasmic genes in maize. In thefirst step of the method, Tripsacura dactyloides (female) is crossed byZea diploperennis (male) by controlled pollination technique. Theresulting intergeneric hybrid derived in step 1 is fully fertile andcross-fertile with maize. It is characterized by its utility as agenetic bridge to transfer Tripsacura genes into corn.

In another embodiment of the invention, the intergeneric (Tripsacumdactyloides X Zea diploperennis) hybrid plant derived from step 1,referred to as Tripsacorn, and maize are crossed by controlledpollination. This invention relates to the hybrid seed, the hybrid plantproduced by the seed and/or tissue culture, variants, mutants, andmodifications of Tripsacorn, of (maize X Tripsacorn) and of (TripsacornX maize).

In another embodiment of the invention, there is provided plants andplant tissues produced by the method of crossing maize (female) byTripsacorn (male) which exhibit resistance to rootworm.

In another embodiment of the invention, there is provided plants andplant tissues produced by the method of crossing maize (female) byTripsacorn (male) which exhibit tolerance to drought.

In another embodiment of the invention, there is provided plants andplant tissues produced by the method of crossing maize (female) byTripsacorn (male) which exhibit enhanced resistance to disease.

In another embodiment of the invention, there is provided plants andplant tissues produced by the method of crossing maize (female) byTripsacorn (male) which exhibit enhanced resistance to insect pests.

In another embodiment of the invention, there is provided plants andplant tissues produced by the method of crossing maize (female) byTripsacorn (male) which exhibit resistance to lodging.

For the purposes of this application, the following terms are defined toprovide a clear and consistent description of the invention.

Allopolyploid. An individual with two or more chromosome sets.

Amphiploid. An individual with two or more genomes derived fromdifferent species.

Antibiosis. Antibiosis refers to the plant's ability to adversely effectthe insect pest, for example by producing a toxic substance.

Antixenosis. Antixenosis refers to the plant's ability to detract theinsect pest away from the plant, for example by producing a deterrentsubstance.

Hybrid plant. An individual plant produced by crossing two parents ofdifferent genotypes.

Polyploid. An individual with some variation in normal diploidchromosome number.

Root Lodging. Root lodging is indicated when plants lean from theverticle axis an at angle ≧30°.

Tolerance. Tolerance is indicated when a plant sustains rootworm damagebut is still able to grow in spite of damage.

DETAILED DESCRIPTION OF INVENTION

The method of the invention is performed by crossing Tripsacumdactyloides and Zea diploperennis. The crosses are performed usingstandard plant breeding techniques for controlled pollinations known inthe art.

Thus, the present invention provides a method of producing hybrid plantseeds comprising the steps of (a) crossing a Tripsacura species (e.g.Tripsacura dactyloides) female parent with a Zea species (e.g. Zeadiploperennis) male parent to produce seed; then (b) harvesting the seedproduced.

This method produces a hybrid seed and a hybrid plant, from which tissuecultures can be made. Additionally, pollen produced by the hybrid plantcan be collected.

The term "plant" as used in this application refers to the whole plantas well as its component parts, e.g., flowers, roots, fruits, andrhizomes.

The present invention further provides a method of producing hybrid cornseed comprising the steps of (a) crossing a Tripsacura dactyloidesfemale parent with a Zea diploperennis male parent to produce(Tripsacura dactyloides X Zea diploperennis) hybrid seed; then (b)growing a (Tripsacura dactyloides X Zea diploperennis) hybrid plant fromsaid seed to maturity; then (c) crossing said (Tripsacura dactyloides XZea diploperennis) hybrid plant with maize to produce seed and (d)harvesting the seed produced.

This method results in the production of hybrid corn seed and hybridcorn plants, from which tissue cultures can be made. One marked benefitof the present invention is the production of hybrid corn plants whichexhibit enhanced resistance to corn rootworm.

Plant breeding techniques and tissue culture techniques as describedhereinare known, and may be carried out in the manner known to thoseskilled in the art. See, for example, U.S. Pat. No. 4,737,596 to Seifertet al. entitled "Hybrid Corn Plant and Seed"; U.S. Pat. No. 5,059,745 toFoley entitled "Hybrid Corn Line LH195"; U.S. Pat. No. 4,545,146 toDavis entitled "Route to Hybrid Soybean Production"; U.S. Pat. No.4,627,192 to Fick entitled "Sunflower Products and Methods for theirProduction", and U.S. Pat. Nos. 4,837,152 and 4,684,612 entitled"Process for Regenerating Soybeans" Applicant specifically intends thatthe disclosure of all U.S. patent applications cited herein beincorporated herein by reference.

In Tripsacum inflorescences, the staminate (i.e. male) flowers andpistillate (i.e. female) flowers are produced on a single spike with themale flowers subtended by the female. When Tripsacum sends out theinflorescence, the staminate flowers are broken off leaving only thefemale flowers on the spike which is then covered with a pollinatingbag, i.e. standard ear shoot bag for maize, to protect them fromcontamination by unwanted pollen. Diploperennis male and female flowersoccur on separate parts of the plant. The staminate flowers are borne inthe tasselwhich emerges at the apex of the culm; whereas, the pistillateflowers occur in single-rowed spikes borne on lateral branches of theculm. When diploperennis produces its tassels, they are covered with apollinating bag. When they start shedding pollen, the bag is removed andpollen taken to pollinate the Tripsacum plants. At that time, the bagscovering the Tripsacum pistillate flowers are removed and thediploperennis pollen shaken out of the bag onto the silks. The Tripsacuminflorescence is covered again with a pollinating bag immediately afterpollination and thebag is stapled so that it remains on the spike untilthe seed has matured. Upon maturity, approximately 45 days later, theseed is harvested. Once mature seed from the cross has been obtained, itis germinated on moist filter paper in a petri dish in the dark. Whenthe seed starts to germinate, it is transferred to potting soil in apot. The plants are grown in the greenhouse or outdoors. Controlledcrosses are best made in agreenhouse where plants are kept isolated toprevent cross contamination and there is no problem with bags beingdamaged by weather conditions.

This method may alternatively be used to cross the plants withdiploperennis as the female parent. In this embodiment, all the tassels,i.e. male flowers, are removed from the diploperennis plant as soon asthey emerge and the ears, i.e. female flowers, are covered withpollinating bags. Rather than removing Tripsacum male flowers, thespikes are left intact and covered with a pollinating bag to collectTripsacum pollen. The pollen is applied to the diploperennis ears whichare then immediately covered with a pollinating bag that is wellfastened with staples to ensure it remains sealed until the seed hasmatured, approximately 45 days after pollination when the seed isharvested.

Next, when (Tripsacum X diploperennis) starts to flower, the same stepsdescribed above are used to cross the hybrid with maize. To cross ontomaize, as soon as the maize plants begin to produce ears, before thesilksemerge, the ears are covered with an ear shoot bag. Pollencollected from (Tripsacum X diploperennis) is applied to silks of themaize ears. The ears are then covered again with an ear shoot bag and alarge pollinating bag which is wrapped around the culm and secured witha staple. The ears remain covered until they reach maturity, severalweeks later when the ears are harvested.

Plants grown from all crosses described above are male and femalefertile and are cross-fertile with each other.

The principles and techniques used in breeding insect and diseaseresistance are basically the same. First, sources of resistance genesmustbe located. Secondly, genes for resistance must be transferred intoadaptedvarieties by hybridization procedures, and thirdly, thosevarieties must beexposed to the insect pest or disease under natural orartificially inducedconditions in order to distinguish resistant strainsfrom susceptible strains. The mode of inheritance of resistance may besimple and involve only one to two major genes. Though in most casesresistance is dominant, it may be dominant or recessive. Inheritance ofresistance also may be more complex with numerous genes affecting thehost-parasite relationship.Plant breeders test for resistance byexperimental inoculation of plants grown in the field and/or thegreenhouse. In testing for rootworm resistance, artificially rearedinsects are transferred to plants grown inthe field or a greenhouse, orto newly germinated seedlings in petri dishes. The infected plants areobserved and evaluated according to specific criteria for a particularpest. In looking for rootworm resistance, criteria for evaluationinclude observations of plant lodging and scoring of root damage by astandardized scale.

Tripsacum X diploperennis

A detailed description of the plants obtained from (Tripsacum Xdiploperennis) is outlined below.

Origin: Seedling

Parentage:

Seed parent.-Tripsacum dactyloides

Source: Established clone at Indiana University, Bloomington, Ind.

Pollen partent.-Zer diploperennis

Source: Jalisco, Mexico (Reference Iltis et al., 1979)

Classification: Botanic--Zea indiana (proposed).

Cytology: Diploid chromosome number determined from root tips rangedfrom 2n=18 to 2n=20.

Habit: Essentially erect; as many as 35 primary culms, usual numberabout 15.

Duration:

Perennial.--Sends out shoots from rhizomes. Plant will freeze at wintertemperatures below 28° F., but new growth is produced in spring afterwinter temperatures of 0° F. Culm:

Height.--Up to two meters: slender, simple with occasional branchingfrom the nodes of the culm; glabrous; oval in cross section; diameter1-1.2 cm.

Nodes.--glabrous, aerial roots develop at nodes along culm.

Sheath.--tightly closed enwrapping the culm, margins not united;glabrous;turns rose red (Pantone #18-1852) when exposed to sun,otherwise green; rose red (Pantone #18-1852, ciliate auricles at summitmargins.

Ligule.--present on adaxial side of leaf at junction of blade andsheath; length: 4 nun; membranaceous, irregular edge.

Leaf blade: Alternate; distichous; sheathing base; parallel veined;narrowly linear, flat, thin.

Length.--47-56 cm. Width: 1.5-5.0 cm.

Entire margin.--Rose red (Pantone #18-1852), serrulate.

Midrib.--White (Pantone #12-5202).

Adaxial surface.--Sparsely hirsute.

Abaxial surface.--Glabrous except sparsely hirsute along midrib.

Prominent parallel veins.--5 per 1 cm width.

Inflorescence

Blooming period: Twice annually in the greenhouse for approximately onemonth beginning in late April and late October in Tennessee, NorthCarolina and Mississippi.

Monoecious: Separate male and female flowers on the same plant;variable.

Staminate flowers: May be of two types: one inflorescence type borne aspaired spikelets on a slender rachis forming 3-7 racemes arranged in apanicle, the "tassel", at the summit of the culm. Alternatively,staminatespikelets may be borne on a single spike above the pistillateflowers.

Length.--6-12 cm.

Axis.--stiff, continuous, ascending.

Spikelet: Two-flowered, one sessile, one pedicled; laterally compressedawnless, attenuate with red (Pantone #19-1860) tip and red (Pantone#19-1860) band at base; Length: 11 mm;

Width: 3 mm. In pairs on one side of a persistent central axis.

Pedicel length.--3 mm.

Glumes.--Outer glume: cartilaginous, tapering to an acute tip, ciliate,flat, several nerved, margins involute, fimbriate.

Inner glume: chartaceous.

Pistillate flowers: Borne in leaf axils; spikelets distichouslyarranged; variable.

Styles: pilose with distinct bifurcated tips.

Color: Ranges from pastel parchment (Pantone #11-0603) to light lilac(Pantone #12-2903) to rose red (Pantone #18-1852).

Length: 100 mm.

One type of pistillate flower consists of a single rowed spike of 4 to 6triangular caryopses in hard, shell-like fruitcases enclosed in a singleleaf sheath; caryopses disarticulate upon maturity.

Length: 7.5 mm; Width: 5 mm.

Colors range from solid to variegated combinations of thefollowing:White (Pantone #11-0602), gray (Pantone #16-1107) , tobaccobrown (Pantone #17-1327), brown (Pantone #19-1121) , dark brown (Pantone#19-1020).

Alternatively, spikelets paired and partially enclosed in stiff, brownspeckled glumes; caryopses rounded and imbricate; Spikes enclosed insingle or multiple leaf sheaths. Caryopses do not disarticulate uponmaturity;

Length: 5 mm; Width: 5 mm.

Color variegated combinations of the following: dark brown (Pantone#19-1217, brown (Pantone #18-1154), beige (Pantone 15-1225), light beige(Pantone #13-1018).

Fruit: Five to ten ears per culm per blooming period; flowers areproduced twice a year under greenhouse conditions; some plants mayproduce approximately 150 ears twice annually.

Maturity: 45 days following fertilization.

Ear (Husked Ear Data Unless Stated Otherwise)

Length: About 43 mm.

Midpoint diameter: About 6.7 mm.

Weight: 0.5 gm.

Kernel rows: 2 (rarely 3-4)

Silk color (exposed at silking stage): light lilac (Pantone #12-2903) torose red (Pantone #18-1852).

Husked color: Cob kernels are embedded in the rachis segments, some ofwhich disarticulate upon maturity. These segments are brownish gray andare the hard, bony fruitcases enclosing the kernels.

Kernel color: beige (Pantone #14-1122) shading to golden beige (Pantone#16-1336).

Husked extension (harvest stage): About 1 cm.

Shank: About 6.5 cm.

Taper: Slight.

Position in dry husk stage: Upright.

Drying time (unhusked ear): About 2-3 days.

Kernel (Dried)

Type I: Angular caryopses in hard, shell-like fruitcases, disarticulateupon maturity:

Size (from midpoint): Length about 0.8 mm, width about 0.5 mm, thicknessabout 0.4 mm.

Shape: Trapezoidal

Colors range from solid to variegated combinations of the following:white (Pantone #11-0602), gray (Pantone #16-1107), tobacco brown(Pantone #17-1327), brown (Pantone #19-1121) , dark brown (Pantone#19-1020)

Weight 20 seeds (unsized samples): 1.3 gm.

Type II: Paired caryopses partially enclosed in endurated glumes forminga cob, upon maturity do not disarticulate:

Size (from midpoint): Length about 3.9 mm, width about 2.8 mm, thicknessabout 2.7 mm.

Shape grade (% round): 100% round (tip pointed).

Pericarp color: beige (Pantone #14-1122) shading to

golden beige (Pantone #16-1336).

Aleurone color: Clear.

Endosperm color: White (Pantone #11-0601).

Endosperm type: Pop.

Weight 20 seeds (unsized samples): About 0.4 gin.

Cob

Diameter at midpoint: 5.3 to 8.7 mm.

Strength: Variable.

Color: Smoke (Pantone #12-0704).

Alicole: Length: About 6.6 mm. External width: 7.0 mm. Internal width:5.0 mm. External length: 5.5 mm. Internal length: 5.0 mm. Thickness:Approximately 4.5 mm. Depth: 2.9 mm.

Cupule: Overhang: About 0.6 mm. Wing height: 4.1 mm. Left wing width:1.0 mm. Right wing width: 1.3 mm. Lower glume length: 5.9 mm. Lowerglume width: ˜3.0 mm. Lower glume angle: ˜20°Glume cushion width: 5.4mm. Glume cushion height: 1.8 mm. Sessile thickness: 0.3 mm. Cupulepubescence: sparse, short hairs. Color: Buff (Pantone #13-1024).

Comparative Parental Characteristics

Duration: Z. diploperennis does not survive temperatures belowapproximately 24° F. T. dactyloides is a true perennial and produces newgrowth every year surviving temperatures are below 0° F.

Leaf blade: Zea diploperennis round in cross section; diam. 1 cm.Tripsacumdactyloides oval in cross section; diam. 1.3 cm.

Leaf blade: Z. diploperennis. Width 1-2 cm; margins pink serrulate frommidsection of blade to tip; adaxial surface: sparsely hirsute; prominentveins: 6 per 1 cm width. T. dactyloides. Width: 1 cm; margins whiteserrulate along entire blade; Adaxial surface: hirsute; prominent veins:12 per 1 cm.

Blooming period: Z. diploperennis twice a year in the greenhouse, end ofMarch and end of September for about a month. T. dactyloidescontinuously from May to October.

Staminate flowers: Z. diploperennis borne in tassel at summit of culm.T. dactyloides staminate flowers borne above pistillate flowers insingle spike.

Pistillate flowers: Z. diploperennis caryopsis triangular-trapezoidal inhard bony fruitcases; Length: 8 mm; Width: 4-5 mm; Color: black (Pantone#19-0303), dark brown (Pantone #19-1020) or mottled black-brown. T.dactyloides caryopsis trapezoidal in hard, bony fruitcase; Length: 6-10mm; Width: 6 mm. Color: pale brown (Pantone #17-1137) or buff (Pantone#13-1024).

Maize X Tripsacorn

(Maize X Tripsacorn) plants look basically like maize. One differencewhen comparing these plants to maize controls is that they are shorter,have stronger stalks and are not susceptible to lodging. The ears looklike maize and are equal in weight to maize ears. However, the kernelstend to be larger in size than kernels of maize controls. The plantsproduced by (maize X Tripsacorn) do not show as many signs ofinfestation by insects or disease as maize controls. Noticeableresistance to aphids and white flies has been observed on plants grownin the greenhouse and enhanced resistance to corn earworm and ear andkernel rot has been observed in plants grown in the field. Laboratorybioassays have shown enhanced resistance to corn rootworm. Whensubjected to dry conditions, (maize X Tripsacorn) plants do not exhibitsigns of wilting and drought stress to the same extent as maizecontrols.

EXAMPLES Bioassays for Determining Rootworm Resistance in Maize

Two types of bioassays, in petri dishes and in pots, were conducted todetermine if Tripsacorn could impart rootworm resistance to maize. Forthebioassays, 1,000 non-diapausing western corn rootworm eggs in soilwere shipped from French Agricultural Research, Inc. , Lamberton, Minn.,to Durham, N.C., under U.S. Department of Agriculture permit number922762. Plants were infested with newly hatched first instar larvae ofwestern corn rootworm, Diabrotica virgifera. The larvae were transferredto test containers by lifting with a small paint brush. Two separatepetri dish bioassays and three pot bioassays were performed.

For the bioassays, seed from Tripsacorn crossed to four diverse types ofmaize was used. The four types included: a commercial hybrid corn seedFunk's G4522; two inbred lines, B73 and W64A; a native Mexican race,Zapalote Chico, classified as a prehistoric mestizo indicatingderivation from ancient indigenous races. Other plants infested withcorn rootworm included (G4522 X Sun Dance), Tripsacum, Tripsacorn andmaize controls.

Petri Dish Bioassays

Petri dish bioassays were employed to screen for antibiosis versusantixenosis by observing whether larvae remained on the roots, ate themand survived or died; or whether larvae moved away from the roots. Ifthere is an antibiotic effect, evidence for eating and dead larvae canbe seen; if there is an antixenotic effect, larvae can be observedtrying to leave the dish. For these tests, 10 grams of top soil sievedthrough a 1 mm mesh screen was placed in a petri dish with 3 to 5freshly germinated seedlings or, in the case of Tripsacum, with a smallclonal piece of plantwith young roots, and kept moist. The rims of eachdish were ringed with petroleum jelly to monitor for any larvae tryingto leave the dish. Up to a total of 50 larvae were added to each dishover a three day period. Eachtreated dish was observed for several daysunder a dissecting microscope at60X magnification and behavior recorded.

The plants used in the petri dish bioassays and observed results aresummarized in Table I. In all cases, larvae remained on or near theroots,seed and cotyledons or in the soil. There was no indication oflarvae trying to exit the petri dishes and thus, it is concluded noevidence for antixenosis. Tripsacum, Tripsacorn, (B73 X Tripsacorn),(W64A X Tripsacorn), (G4522 X Tripsacorn) and (G4522 X Sun Dance) didnot show anysigns that the roots produce a substance that is a deterrentto the insects. Larvae feeding was observed in all cases and severity ofroot damage rated by the Hills and Peters scale. Evidence for antibiosisand tolerance was indicated with Tripsacorn and the hybrids between cornand Tripsacorn tested; whereas, there was no evidence for antibiosis ortolerance with the corn and (maize X Sun Dance) materials tested.

                  TABLE I                                                         ______________________________________                                        RESULTS OF PETRI DISH BIOASSAYS                                                          No. of Larvae                                                                           Observations/Comments                                    ______________________________________                                        Bioassay #1                                                                   Tripsacum    50          Larvae stay on root,                                                          some feeding but                                                              virtually no damage to                                                        roots, larvae not visible                                                     after a couple of days                               Tripsacorn   50          Some feeding, little root                                                     damage                                               B73 X Tripsacorn                                                                           50          Some feeding, little root                                                     damage, plants continue                                                       to grow                                              G4522 X Tripsacorn                                                                         50          Some feeding, little root                                                     damage, plants continue                                                       to grow                                              G4533 X Sun Dance                                                                          50          Extensive feeding, plants                                                     died                                                 Corn control 50          Extensive feeding, plants                                                     died                                                 Bioassay #2                                                                   Tripsacorn   20          Light feeding, some dead                                                      larvae                                               Corn control (W64A)                                                                        45          Extensive feeding, plants                                                     died                                                 W64A X Tripsacorn                                                                          45          Feeding on roots, seed                                                        and cotyledons, some                                                          dead larvae                                          ______________________________________                                    

Pot Bioassays

Plants grown in pots were used to screen for evidence of toleranceand/or antibiosis. Lodging is seen in plants that are susceptible torootworm damage; whereas, plants that remain upright and healthy whenexposed to rootworms are indicative of tolerance and antibiosis. Rootdamage was observed and scored according to the Hills and Peters (1971)rating scale of 1-6 that is widely used in the corn belt to evaluateroot damage. The criteria for rating are as follows:

1. No damage or only a few minor feeding scars

2. Feeding scars evident but no roots eaten off to within 1 1/2 inch oftheplant

3. Several roots eaten off to within 11/2 inch of the plant but neverthe equivalent of an entire node of roots destroyed

4. One root node completely destroyed

5. Two root nodes completely destroyed

6. Three or more root nodes destroyed

When a bioassay was complete, two to three plants were removed from thepots, soaked in water then rinsed with a gentle water spray to clean theroots, then observed under a dissecting microscope for scoring. Thescore reported is the mean calculated from the total scores of plants ineach category. Tolerant plants may suffer root damage but are capable ofregrowth and degrees of plant recovery. Well developed secondary rootsystems are often capable of compensatory growth from damaged crownroots.

In the first pot bioassay, 3 to 5 seedlings (approximately 1 week old),or in the case of Tripsacum a small clone with young roots, were plantedin potting soil in 10-ounce containers and were grown indoors underartificial grow lights. A total of 70 larvae were added to eachcontainer over a two day period and plants were observed for 11 days.

In the second pot bioassay, ≦10 day old seedlings were planted inpotting soil in 3 inch peat pots and grown indoors under artificial growlights. A total of 30 larvae were added to each pot over a three dayperiod. Although most plants were dead within one week, observation oftheones that survived extended over two weeks before plants weresacrificed for root evaluation. For each type, there were a minimum oftwo plants, and in most cases there were four plants.

In the third pot bioassay, the plants were 11 to 14 days old atinfestationand they were grown on a porch under natural sunlight. Atotal of 30 larvaewere added to each pot over two days. They wereobserved for 11 days beforesacrificing plants to score root damage.

The plants used in the pot bioassays and observed results are summarizedinTable II. The results indicate that (maize X Tripsacorn) plants aredefinitely more resistant to corn rootworm than corn controls and (maizeXSun Danse). The mechanisms indicated for resistance inherited fromTripsacorn are antibiosis and tolerance. All the plants sustained someinjury to the roots. Lodging in the corn controls and (maize X SunDance) plants was ≧45° and rating on the Hills and Peters scale rangedfrom 5 to 6. The corn X Tripsacorn plants remained upright and appearedhealthy, but did sustain root damage. There was good development ofsecondary roots from the damaged crown showing the capability forcompensatory growth in all the (maize X Tripsacorn) plants.

                                      TABLE II                                    __________________________________________________________________________    RESULTS OF POT BIOASSAYS                                                                 No. of Larvae                                                                         Duration                                                                           Root Damage*                                                                          Observations/Comments                         __________________________________________________________________________    Bioassay #1                                                                   Tripsacum  70      11 days                                                                            Not recorded                                                                          No sign of damage                             Tripsacorn 70      11 days                                                                            Not recorded                                                                          No sign of damage                             B73 X Tripsacorn                                                                         70      11 days                                                                            Not recorded                                                                          No sign of damage                             G4522 X Tripsacorn                                                                       70      11 days                                                                            Not recorded                                                                          No sign of damage                             G4522 X Sun Dance                                                                        70      11 days                                                                            Not recorded                                                                          Plants died after                                                             6 days                                        Bioassay #2                                                                   Corn control (W64A)                                                                      30      14 days                                                                            5.0     Plants died                                   W64A X Tripsacorn                                                                        30      14 days                                                                            2.0     Plants weakened                               G4522 X Sun Dance                                                                        30      14 days                                                                            5.0     Plants died                                   Bioassay #3                                                                   Corn control                                                                             30      11 days                                                                            4.0     Lodging (≧45°),                 (Zapalote Chico)                leaf damage                                   Zapalote Chico                                                                           30      11 days                                                                            2.0     Minor leaf damage                             X Tripsacorn                                                                  Corn Control (W64A)                                                                      30      11 days                                                                            4.0     Lodging (≧45°)                  W64A X Tripsacorn                                                                        30      11 days                                                                            2.3     Plant upright and                                                             growing                                       G4522 X Sun Dance                                                                        30      11 days                                                                            5.0     Lodging (≧45°),                                                 leaf damage                                   Tripsacorn 30      11 days                                                                            1.0     No evidence of                                                                damage                                        __________________________________________________________________________    *Hills and Peters scale (1971)                                            

DEPOSIT OF SEEDS

Seeds derived from crosses between Tripsacum dactyloides and Zeadiploperennis as described herein were deposited in accordance with theprovisions of the Budapest Treaty with American Type Culture Collection,12301 Parklawn Drive, Rockville, Md. 20852 on Aug. 28, 1992. Theaccessionnumber is ATCC75297.

The present invention is not limited in scope by the seeds deposited,sincethe deposited embodiments are intended as single illustrations ofone aspect of the invention and any seeds, cell lines, plant parts,plants derived from tissue culture or seeds which are functionallyequivalent arewithin the scope of this invention. While the inventionhas been described in detail and with reference to specific embodimentsthereof, it will be apparent to one skilled in the art that changes andmodifications can be made without departing from the spirit and scope ofthe invention in addition to those shown and described herein. Suchmodifications are intended to fall within the scope of the appendedclaims.

I claim:
 1. A method of producing a hybrid plant seed having a diploidchromosome number of between 18 and 20, comprising the steps of:(a)crossing a Tripsacum dacyloides female parent with a Zea diploperennismale parent to produce a seed; then (b) harvesting said seed produced in(a); wherein said seed has all of the identifying characteristics ofATCC 75297, and wherein a plant grown from said seed is a fertilehybrid.
 2. Seed produced in accordance with the method of claim
 1. 3. Ahybrid plant grown from seed according to claim 2, said plant beingfertile and having a diploid chromosome number of between 18 and
 20. 4.Pollen produced by a plant according to claim
 3. 5. A tissue cultureproduced from a plant according to claim 3, the cells of said tissueculture having a diploid chromosome number of between 18 and
 20. 6. Amethod of producing a hybrid maize seed comprising the steps of:(a)crossing a Tripsacum dactyloides female parent with a Zer diploperennismale parent to produce (Tripsacum dactyloides X Zea diploperennis)hybrid seed; then (b) growing a (Tripsacum dactyloides X ZerDiploperennis) hybrid plant from said seed to maturity, then (c)crossing said (Tripsacum dactyloides X Zer diploperennis) hybrid plantwith Zea mays to produce seed; (d) harvesting the seed produced in (c),wherein said seed produced in (a) all of has the identifyingcharacteristics of ATCC 75297, and wherein said seed produced in (c)germinates into a plant having resistance to corn rootworm (Diabroticavirgifera).
 7. Hybrid maize seed produced in accordance with the methodof claim
 6. 8. Hybrid maize plants grown from said seed of claim 7 whichmaize exhibits resistance to corn rootworm.
 9. Hybrid maize plants grownfrom said seed of claim 7 which maize exhibits resistance to lodging.10. Plants produced by in vitro propagation of said hybrid maize plantsof claim 8, wherein said plants produced by in vitro propagation haveresistance to corn rootworm (Diabrotica virgifera).
 11. The method ofclaim 1, wherein said seed produced is ATCC
 75297. 12. The method ofclaim 6, wherein said hybrid seed produced in step (a) is ATCC 75297.